1. Field of the Invention
The present invention provides methods of producing a surface with enhanced cell-adhesive properties comprising applying a stress to a polymeric matrix. The strained matrix is then modified by grafting a self-assembled monolayer onto the strained matrix, with the self-assembled monolayer comprising at least one exposed functional group. At least one cell-adhesive molecule can then be coupled to the at least one exposed functional group on the self-assembled monolayer to produce a surface with enhanced cell-adhesive properties.
2. Background of Invention
Cell and tissue culture in vitro has been routinely practiced in many areas of the biological and medical arts. However, many primary cells derived from human tissues are not capable of being supported for survival, proliferation, or differentiation in vitro using the conventional tissue culture techniques. This inability has limited the application of cell culture in areas such as cellular therapies and drug screening because of a gap between the in vivo state of the cells where they are organized into three-dimensional tissues that are constantly subjected to mechanical stresses and deformations and the in vitro state where the cells try to recapitulate the in vivo state on a static two-dimensional surface. Bioengineered tissue/scaffold constructs have emerged that are starting to bridge the gap, addressing the three-dimensional aspects of living tissues. The importance of mechanical stresses on growth and differentiation of cells in living tissues has also been recognized and accordingly led to the development of in vitro cell culture systems allowing stresses to be applied.
For example, Flexcell culture systems from Flexcell International Corporation are able to apply tensile, compressive or shear stresses to cultured cells. (See, for example, U.S. Pat. Nos. 4,789,601, 4,822,741, 4,839,280, 6,037,141, 6,048,723, and 6,218,178.) U.S. Pat. No. 6,057,150 discloses that the application of a biaxial strain to an elastic membrane that may be coated with extracellular matrix proteins and covered with cultured cells. U.S. Pat. No. 6,107,081 discloses another system in which a unidirectional cell stretching device comprising an elastic strip is coated with an extracellular matrix on which cells are cultured and stretched.
The central component of the above-mentioned cell culture systems that allows for the application of mechanical stresses is a flexible substrate that can be deformed easily and in a controlled manner, and which also supports cell adhesion and growth comparable to conventional cell culture substrates. Silicones, such as poly(dimethyl siloxane) (PDMS), are particularly suitable for this application because they are not only highly flexible but also provide optical clarity that allows microscopic observation of the cell cultures. However, PDMS surfaces do not support cell adhesion, and must to be modified before they can be used as cell culture substrates. Surface modification of silicone surfaces is complex and requires the introduction of functional groups that either by themselves allow for cell attachment or that are available for subsequent coupling of cell adhesion promoting ligands, for example, —NH2 or —COOH groups.
Radio-frequency (RF-) plasma treatment can be used to introduce functional groups into the polymer surfaces. For example, an oxygen plasma treatment can be used to turn a polystyrene surface that will support only limited cell adhesion into a surface that will readily support cell attachment and growth. However, plasma treated surfaces gradually degrade over time because of the migration of the polar functional groups away from the surface into the polymer bulk. This effect is increased in PDMS because of the high chain mobility caused by the low glass transition temperature (below room temperature) of PDMS. Murakami et al., Journal of Colloid and Interface Science 200: 192(1998).
Several alternative derivatization methods to create silicone-based cell culture substrates are disclosed in U.S. Pat. Nos. 4,789,601 and 4,822,741. Exposure of cured or uncured silicone to a Bunsen burner flame leads to the incorporation of elemental carbon particles that were found to increase biocompatibility. Amination induced by HCl treatment followed by exposure to NH4OH or ammonia vapor was also found to increase the biocompatibility of that surface. Amination followed by peptidization (covalent coupling of a peptide to glutaraldehyde activated aminated surface) presents yet another way to improve the biocompatibility of silicone. In yet another method, amination is achieved by co-curing a polyorganosiloxane with a primary amine- or carboxyl-containing silane or siloxane.
A drawback of these conventional surface modification techniques of silicones for cell culture application is the limited density of functional groups that are imposed on the surface, thus leading to limited cell attachments.
Genzer et al. have previously reported a surface derivatization method called mechanically assembled monolayers (MAMs) on elastomeric silicon substrates rendering the surfaces either super-hydrophobic (Genzer et al., Science, 290: 2130 (2000)), covalently coupled with dense polymer brushes (Wu et al., Macromolecules, 34: 684 (2001)), or expressing a chemical gradient on the surface (Efimenko et al., Advanced Materials, 13: 1560 (2001)). However, it is not clear whether the elastomeric silicon substrates modified by this surface derivatization method is feasible for cell culture and cell growth. Thus, there is a need in the art for improved substrates and methods for culturing cells that support cell adhesion, growth and differentiation and that allow the application of mechanical stress to the cells.